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Short Communications |
From the Unit of Molecular Pathology,*
International
Agency for Research on Cancer (IARC), Lyon, France; the First
Department of Surgery, Yamanashi University of Medical
School,
Yamanashi, Japan; and the Department
of Pathology,
Institute of Clinical
Pathology, University Hospital Zürich, Zürich, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, representing 4% of all malignant tumors, and is the seventh most frequent carcinoma in males and the ninth most frequent carcinoma in females.10-12 Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, exposure to aflatoxin B1, and excessive intake of alcohol have been identified as major risk factors.10,13,14 HCCs associated with HBV infection are most frequent in Southeast Asia and sub-Saharan Africa,15 whereas HCCs associated with HCV are most prevalent in southern Europe and Japan. In Italy, Spain, and Japan, 50–75% of cases of HCCs are associated with HCV infection.16
Mechanisms underlying hepatocarcinogenesis associated with HBV and HCV infection are not fully understood, except that both HBV and HCV virus infections can lead to chronic infection, causing cirrhosis, which is a well-known precursor of hepatic malignancy.13,17 HBV is a double-stranded DNA virus and has been shown to be integrated into the HCC genome of most patients with serological evidence of HBV infection,10,17 suggesting a direct carcinogenic effect through interaction with transformation-associated genes. Furthermore, the X protein of HBV appears to be a potent transactivator and to interact with p53, possibly interfering with its tumor suppressor activity.18 In contrast, HCV is a single-stranded, positive-sense RNA virus and appears to be a nonintegrating virus.17,19 However, the HCV core protein has some potential direct carcinogenic effects in vitro. HCV core protein transforms rat embryo fibroblasts into a malignant phenotype and suppresses apoptotic cell death in culture.20,21 It may also play an important role in the promotion of cell growth by repressing the transcriptional activity of p53.22 Furthermore, the HCV core protein induces HCCs in transgenic mice.23
Molecular mechanisms underlying the development of HCCs are still
poorly understood. p53 mutations have been found in about
25–45% of HCCs (see the review by Montesano et al).14
HCCs that develop in patients highly exposed to aflatoxin
B1 frequently contain a specific G
T transversion
mutation in codon 249, whereas HCCs from areas with little exposure to
this carcinogen did not contain specific p53
mutations.14
Cyclin D1 gene was amplified 3–16-fold in
five of 45 (11%) HCCs associated with HBV or HCV
infection.24
Activation of c-myc by DNA
amplification was observed in 28 of 77 (36%) HCCs, and this occurred
more frequently in the patients with HBV infection than in those with
HCV infection.25
Reduced expression of E-cadherin due to
methylation at CpG sites around the promoter region and allelic
deletions of the E-cadherin gene itself have been recognized in about
60% of HCCs.26,27
Recently, ß-catenin mutations were
found in 19–23% of etiologically unspecified HCCs,28,29
suggesting a role for the Wnt signaling pathway in the evolution of
HCCs.
The objective of this study was to assess involvement of the Wnt signaling pathway in HCCs associated with HCV infection. We have screened mutations of the ß-catenin and APC genes by SSCP followed by DNA sequencing in 22 HCCs associated with HCV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sixteen HCCs associated with HCV infection were obtained from the
First Department of Surgery, Yamanashi Medical University (Yamanashi,
Japan), and six were from the Department of Pathology, Institute of
Clinical Pathology, University Hospital (Zürich, Switzerland).
HCV serology was performed by second- or third-generation
enzymed-linked immunosorbent assay and tested positive in all cases.
HCV RNA was also tested by standard reverse transcriptase-polymerase
chain reaction (RT-PCR) methods and was positive in all cases. All of
the patients were negative for HBs antigen. The mean age of the
patients was 62.7 ± 6.3 years (range 49–75 years). Seventeen
patients were males and five were females (Table 1)
. HCCs were diagnosed as well,
moderately, or poorly differentiated, according to the WHO
classification30
(Table 1)
. Tumors were fixed in buffered
formalin and embedded in paraffin. Tumor tissues were manually
microdissected, after microscopic identification and labeling. Care was
taken to avoid contamination with normal or cirrhotic liver tissue. DNA
was extracted as described previously.31
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PCR-SSCP analysis was carried out for exon 3 of the ß-catenin
gene, which contains the four potential GSK-3ß phosphorylation
sites,29
using the following primers:
5'-ATGGAACCAGACAGAAAAG-3' (nt 254–272, sense) and
5'-TACAGGACTTGGGAGGTATC-3' (nt 386–405, antisense). PCR was carried
out in a total volume of 10 µl, consisting of 2 µl of DNA solution,
0.5 U of Taq DNA polymerase (Sigma, St. Louis, MO), 0.5
µCi of [
-33P]dCTP (ICN Biomedicals, Costa Mesa, CA;
specific activity, 3000 Ci/mmol), 1.5 mmol/L MgCl2, 0.2
mmol/L of each deoxynucleoside triphosphate (dNTP), 0.4 µmol/L of
both sense and antisense primers, 10 mmol/L Tris-HCl (pH 8.3), and 50
mmol/L KCl in the RoboCycler Gradient 96 (Stratagene, La Jolla, CA)
with an initial step of denaturation at 95°C for 5 minutes, followed
by 35 cycles of denaturation at 95°C for 1 minute, annealing at
53°C for 1 minute, polymerization at 72°C for 1 minute, and then a
final extension of 5 minutes at 72°C. Five microliters of PCR
products was mixed with 12.5 µl loading buffer (95% formamide, 20
mmol/L EDTA, 0.05% xylene cyanol and bromophenol blue), denatured at
95°C for 10 minutes, and quenched on ice. Four microliters of the
above mixture was run on a 6% polyacrylamide nondenaturing gel
containing 8% glycerol, at 4 W for 14 hours at room temperature,
and/or on a 6% polyacrylamide nondenaturing gel containing 6%
glycerol, at 40 W for 3.5 hours, with cooling by fan. Gels were dried
at 80°C and autoradiographed for 12–48 hours.
Samples that showed mobility shifts in SSCP analysis were further
analyzed by direct DNA sequencing. Two samples that did not show
mobility shifts but showed nuclear accumulation of ß-catenin protein
by immunohistochemistry were also sequenced. PCR amplification was
carried out as described above in the absence of
[-33P]dCTP. Five microliters of PCR products was
digested with 1 U of shrimp alkaline phosphatase and 5 U of exonuclease
I at 37°C for 15 minutes. After inactivation of these enzymes at
80°C for 15 minutes, 15 pmol primer (the same primers for PCR) and 2
µl of 5x sequenase buffer (200 mmol/L Tris-HCl, pH 7.5, 100 mmol/L
MgCl2, 250 mmol/L NaCl) were added. The template-primer
mixture was heated at 100°C for 5 minutes and then placed in ice-cold
water. Dithiothreitol (0.1 mol/L), 3 U Sequenase, version 2.0 (USB,
Cleveland, OH), and 0.5 µCi [-33P]dATP or
[-33P]dCTP were added to samples, which were then
divided into four wells containing each termination mixture. Samples
were incubated at 42°C for 6 minutes and mixed with 5 µl stop
solution (USB). After being heated at 80°C for 3 minutes, samples
were loaded onto a 6% polyacrylamide/7 mol/L urea gel and run at 70 W
for 1.5–3 hours. Gels were dried at 80°C and autoradiographed for
12–48 hours.
SSCP Analysis for APC Mutations
Mutations in codons 1255–1513 in exon 15 of the APC
gene, corresponding to the mutation cluster region (MCR), were screened
by SSCP analyses in 22 HCCs. This region covers about two-thirds of all
APC somatic mutations in colon tumors in FAP and
non-FAP patients.32-34
Three pairs of primers were used
to amplify the following overlapping fragments: for codons 1255–1363
(fragment 15A), 5'-AACCAAGAAACAATACAGA-3' and
5'-CACTTTTGGAGGGAGATTT-3'; for codons 1342–1433 (fragment 15B),
5'-AGAATCAGCCAGGCACAAAG-3' and 5'-GCTTGGTGGCATGGTTTGT-3'; for codons
1410–1513 (fragment 15C), 5'-GCAGTGGAATGGTAAGTGG-3' and
5'-TCATCGAGGCTCAGAGCA-3'. Prescreening for mutations by PCR-SSCP
analysis in this region was performed in a total volume of 10 µl,
consisting of 1 µl of DNA solution, 0.5 U of Taq DNA
polymerase (Sigma), 0.5 µCi of [-33P]dCTP (ICN
Biomedicals; specific activity, 3000 Ci/mmol), 2.0 mmol/L (for fragment
15A) or 1.5 mmol/L (for fragments 15B and C) MgCl2, 0.2
mmol/L of each dNTP, 0.4 µmol/L of both sense and antisense primers,
10 mmol/L Tris-HCl (pH 8.3), and 50 mmol/L KCl in the RoboCycler
Gradient 96 (Stratagene, La Jolla, CA), with an initial step of
denaturation at 95°C for 5 minutes, followed by 40 cycles of
denaturation at 95°C for 60 seconds; annealing at 47°C (for
fragment 15A), 57°C (for segment 15B), or 55°C (for segment 15C)
for 70 seconds; polymerization at 72°C for 70 seconds; and a final
extension of 5 minutes at 72°C. After amplification, 5 µl of PCR
products was mixed with 12.5 µl loading buffer (95% formamide, 20
mmol/L EDTA, 0.05% xylene cyanol, and bromophenol blue), denatured at
95°C for 10 minutes, and quenched on ice. Four microliters of this
mixture was run on a 6% polyacrylamide nondenaturing gel containing
6% glycerol at 40 W for 5 hours with cooling by fan. Gels were dried
at 80°C and autoradiographed for 12–48 hours.
Samples that showed mobility shifts in the SSCP analysis were further analyzed by direct DNA sequencing. The PCR products were sequenced using the same primers for PCR and with the same protocol as described above for ß-catenin mutations.
ß-Catenin Immunohistochemistry
Twenty-two cases of HCCs and their respective nontumorous liver tissue adjacent to carcinomas were subjected to immunohistochemical analysis of ß-catenin expression. The sections were deparaffinized in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase in methanol for 30 minutes at room temperature. For antigen retrieval, the sections were microwaved in antigen unmasking solution (Vector Laboratories, Burlingame, CA) three times for 5 minutes. After incubation with 5% skimmed milk for 1 hour at room temperature, the sections were incubated with the primary antibody against ß-catenin (Transduction Laboratories, Lexington, KY; 1:1000–2000) overnight at 4°C. The reaction was visualized with a Vectastein Elite ABC kit (Vector Laboratories) and 3,3'-diaminobenzidine solution (Vector Laboratories). The sections were then counterstained with hematoxylin. Formalin-fixed, paraffin-embedded sections of human colon were used as positive controls. Sections without primary antibody served as negative controls.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SSCP followed by direct DNA sequencing revealed that nine of 22
(41%) HCCs contained a ß-catenin mutation. All mutations were single
nucleotide substitutions occurring at different putative
phosphorylation sites of serine/theronine or their contiguous residues
in exon 3: three at codon 32, one at codon 33, three at codon 37, one
at codon 41, and two at codon 45 (Table 1
, Figure 1
). In all cases, a wild-type base was
also detectable. Except for two cases (cases 18 and 20), we also
analyzed adjacent cirrhotic, nontumorous liver. None of the adjacent
nontumorous tissues analyzed contained a ß-catenin mutation.
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, Figure 1
). APC Mutation and Polymorphism
SSCP followed by direct sequencing showed no miscoding mutations
but a polymorphism at codon 1493 (ACGACA, ThrThr) in HCCs
analyzed. A/A homozygote, G/G homozygote, and G/A heterozygote were
observed in 12 (55%), two (9%), and eight (36%) HCCs, respectively.
These frequencies were not significantly different from those in blood
DNAs from 50 healthy individuals from USA (A/A homozygote, 40%; G/G
homozygote, 6%; G/A heterozygote, 54%, P = 0.5615,
Fisher’s exact test).
ß-Catenin Immunohistochemistry
In the nontumorous liver tissue examined, the cytoplasmic membrane
of bile duct epithelial cells showed strong ß-catenin protein
expression, whereas the cytoplasmic membrane of hepatocytes showed weak
or moderate expression (Figure 2A)
.
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. In most cases (10 of 11, 91%),
neoplastic cells with nuclear ß-catenin accumulation were restricted
to the periphery of tumor nodules (Figure 2, B and C)
or were in small
tumor nests detached from large nodules. In one case (case 20), strong
ß-catenin protein expression was observed in the nuclei and cytoplasm
of neoplastic hepatocytes, diffusely throughout the tumor. Most
neoplastic cells with nuclear ß-catenin protein accumulation also
showed ß-catenin protein expression in the cytoplasmic membrane. Paraffin sections of normal human colon epithelial cells (positive control) showed strong immunoreactivity to ß-catenin. Sections without primary antibody showed no immunoreactivity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study shows that ß-catenin mutations are frequent (41%) in HCCs associated with HCV infection. This frequency is higher than in previous studies of nonspecified HCCs (19–23%).28,29 It remains to be clarified whether HCCs associated with HCV more frequently contain ß-catenin mutations than HCCs associated with other etiological factors. There is evidence that the frequency of ß-catenin mutations may differ in HCCs induced by different chemical carcinogens in mice. ß-Catenin mutations were found in nine of 24 (37.5%) methylene chloride-induced and 17 of 42 (41%) oxazepam-induced liver tumors, but only in three of 18 (17%) vinyl carbamate-induced liver tumors, one of 18 (6%) TCDD-induced liver tumors, and one of 22 (5%) spontaneous liver tumors in B6C3F1 mice.45
Consistent with previous studies,7 seven of 10 (70%) ß-catenin mutations detected in this study were located at specific serine/threonine residues (codons 33, 37, 41, and 45) of exon 3; this is also consistent with the role of these residues as putative phosphorylation targets of GSK-3ß. Mutations at the contiguous residue of serine 33 may alter the protein structure and limit the access of GSK-3ß, leading to inhibition of phosphorylation of ß-catenin and its degradation through the ubiquitin-proteasome pathway.28
The clonality of HCCs has been studied by several investigators, but
this issue is still controversial.46-49
Based on X-linked
RFLP analysis of the phosphoglycerokinase gene, Aihara et
al48
found that all seven HCCs induced by HCV were
monoclonal. By using the X-chromosome inactivation method, Kawai et
al47
reported that nine of 10 HCCs analyzed were
monoclonal in origin, whereas another one was polyclonal in origin. By
DNA fingerprinting and hepatitis B virus DNA integration pattern
analysis, Sheu46
found that 12 of 18 hepatitis B surface
antigen-positive or negative patients were different in clonality,
suggesting that the majority of HCCs are polyclonal. Of the cases in
this study, two different mutations were detected in two cases (cases
16 and 18), whereas another case (cases 15) showed identical mutations
in three different areas of HCCs (Table 1)
. Taken together, these
results suggest that development of HCC may be polyclonal in some
cases, but intrahepatic metastases may also occur.50
Alternatively, different mutations may be due to genetic heterogeneity
within the tumor.
Mutations of ß-catenin or APC genes result in stabilization of ß-catenin and a significant increase in this protein within the cell. Accumulated ß-catenin may be translocated into the nucleus, where it serves as a transcriptional factor through binding with the Tcf-Lef family.4,7,51 In this study, we showed a correlation between the presence of ß-catenin mutations and nuclear accumulation of ß-catenin protein in HCCs. Nuclear accumulation of ß-catenin was observed in 11 cases; nine of these carried a ß-catenin mutation. The absence of ß-catenin mutations in the other two cases with ß-catenin protein accumulation suggests that there may be other mechanisms leading to ß-catenin accumulation, such as alterations in GSK3ß, axin, or other components of the Wnt signaling pathway. It is of interest to note that, except for one case (case 20), in which tumor cells with nuclear accumulation of ß-catenin were observed diffusely throughout the tumor, the nuclear accumulation of ß-catenin was largely restricted to the periphery of the tumor nests. The mechanism and biological significance of the specific localization of neoplastic cells with nuclear accumulation of ß-catenin remain to be elucidated.
Miscoding APC mutation was not found in any of the HCCs analyzed. Several previous studies suggested that APC may not be alterated in HCCs because the APC locus on chromosome 5q did not show loss of heterozygosity.52-54 Another study55 screened five HCCs by RNase protection assay and revealed no mutations. The present study examined the region that covers the sequence where about 70% of APC somatic mutations have been found in human colon cancers.33,34 Although the possibility of mutations in other regions of the APC gene cannot be excluded, our data suggest that mutation in the ß-catenin, but not the APC gene, is a major causal event in the activation of the Wnt signaling pathway in HCCs associated with HCV infection.
It has been suggested that some polymorphism in the APC gene, rather than altering the function of the encoded protein, may create an unstable tract that is hypermutable, leading to somatic, truncating mutations occurring at adjacent sequences and indirectly causing cancer predisposition.56 Of more than 20 polymorphisms identified to date in the APC gene,57 the I1307K polymorphism has been studied in detail and has been related to a predisposition to colorectal cancer in Ashkenazi Jewish populations.56,58 Little is known about the significance of other APC polymorphisms. In the present study, we did not find the I1307K polymorphism but found the ACG/ACA polymorphism in codon 1493, as previously reported.32 There was no difference in the frequencies of A/A, G/A, and G/G genotypes in APC codon 1493 between HCCs analyzed in this study and blood DNA from healthy individuals.
In summary, we show a high frequency of ß-catenin mutations and focal nuclear accumulation of ß-catenin protein in neoplastic hepatocytes in HCCs associated with HCV infection. These results indicate that activation of the Wnt signaling pathway by ß-catenin mutations is frequently involved in the development of HCCs associated with HCV infection.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Academy of Finland and the Conselho National de Desenvolvimento Científico e Tecnológico, Brazil.
Accepted for publication August 24, 1999.
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